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Figure 6—figure supplement 1 Xenopus tmem79 is required for anterior neural patterning and neural crest formation without affecting the head organizer.
(A) A Tmem79 sense probe as a negative control does not show any hybridization signal in embryos at different stages. (B) Dorsal injection of Tmem79 mRNA induces an enlarged head in >50% embryos and spina bifida in another about 30% embryos. Anterior to right. Also see Supplementary file 1G. (C) Tmem79MO inhibits protein synthesis from the Xenopus Tmem79 (xTmem79) mRNA, but not of the mouse Tmem79 (mT79) mRNA, which is used in rescue experiments. GAPDH is a protein loading control. (D) Usp8MO inhibits protein synthesis from the Xenopus Usp8 (xUsp8) mRNA, but not from a mutant Usp8 (x-mutUsp8) mRNA, in which several nucleotides in xUsp8 5’-UTR targeted by the MO have been altered. (E) Dorsal-animal injection of Tmem79 MO reduces expression of anterior and mid/hind brain markers Bf1, Krox20, and En2, an anterior (cement gland) marker Xag (each seen in the anterior view), and a neuronal marker n-tubulin (see in the dorsal view), examined at stage 16. Co-injection of mTmem79 with Tmem79MO rescues expression of these markers. Also see Supplementary file 1H. (F) Dorsal-animal injection of β-cateninMO or Usp8MO leads to enlarged head formation at the tailbud stage (anterior to right), supporting Usp8 acting positively in Wnt/β-catenin signaling in vivo. Also see Supplementary file 1I. (G) Dorsal-animal injection of Tmem79MO affects neither expression of head organizer genes (Goosecoid, Chordin, Dkk1, and Lim1) nor that of dorsal genes (xnr3 and xnot) at stages 10.5–11. Vegetal views with dorsal to top. Also see Supplementary file 1J. (H) Dorsal-animal injection of Tmem79MO reduces expression of neural crest markers FoxD3 and snail1 examined at stage 16. Also see Supplementary file 1K. |
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Figure 7. Xenopus Tmem79 regulates Frizzled (FZD)/PCP signaling and convergent-extension (CE) cell movements in axial mesoderm. (A to D) Dorsal (axial mesoderm) injection of Tmem79MO causes spina bifida scored at stage 26 (A and B), and a lack of CE movements of axial mesoderm, visualized by a widened (not elongated) xNot-expressing axial tissue and an open blastopore at stage 12 (C and D). Axial mesoderm elongation is rescued by Tmem79 mRNA (C and D). Anterior to right. Also see Supplementary file 1D and E. (E to G) Axial mesoderm induced by activin in animal pole explants exhibits CE movements, visualized by explant elongation and quantified by a length/width (L/W) ratio (E and F). Control explants are round with a L/W ratio of 1 while explants (axial mesoderm induced by activin) elongate with a L/W ratio of 3 (E and F). Explants with Tmem79MO poorly elongate but elongation is rescued by Tmem79 mRNA or Usp8MO (E and F). Also see Supplementary file 1F. Note that Tmem79MO alone or together with Usp8MO does not affect activin-induced axial mesoderm gene expression (G). |
